ABSTRACT According to the World Health Organization 70 million people are visually impaired worldwide due to corneal related diseases and injuries. In the U.S. alone more than 50,000 corneal transplants are performed annually due to lack of alternative treatments. The cornea relies on resident stem cells to sustain vision and efficiently regenerate after injury. Conditions that cause stem cells to deviate from their normal activity lead to corneal disease. Elucidating the identity and mechanism of regulation of corneal stem cells is critical for devising new effective treatments for corneal pathologies. We currently have incomplete knowledge of the stem cell dynamics and regulation in the live cornea. A major roadblock is the inability to visualize directly single cell activity during corneal regeneration to elucidate the precise contribution of stem cells. To overcome this, we have pioneered a novel approach to visualize and track stem cell activity in the intact cornea of live mice by 2- photon microscopy. The overall goal of this project is to implement an integrative approach by combining our live imaging system with state-of-the-art optogenetic and genomic tools to 1) characterize stem cell dynamics, 2) identify intrinsic and extrinsic regulators of stem cell activity and 3) test their requirements for corneal regeneration. In live imaging experiments of the cornea we found that stem and progenitor cells in the self- contained corneal epithelium proliferate and differentiate in topologically diverse patterns during homeostasis and wound healing. We hypothesize that the cornea consists of a heterogeneous population of stem cells with distinct contributions to homeostatic maintenance and injury repair. Aim I of this proposal is designed to resolve the specific contributions of stem cells and their immediate progeny to corneal regeneration and to test their necessity for wound healing. Aim I will also address the role of key molecular signals in regulating the fate of distinct epithelial populations within the cornea. Such signals can potentially be exploited for therapeutic purposes. Aim II will investigate extrinsic regulation of corneal stem cells. The subbasal nerve plexus is a major component of the corneal tissue environment. Our live imaging approach is ideal for studying the functional interactions between the corneal nerves and the epithelium. We have devised in vivo assays for visualizing and manipulating nerve processes in the live cornea. Aim II will test the requirement of the subbasal nerve plexus for epithelial function in homeostasis and wound healing and explore novel mechanisms for corneal nerve regeneration. These data will enable new therapies for patients with Neuropathic Keratitis. This research is innovative because it uses cutting-edge imaging technologies and genetic tools to study stem cells within the natural tissue environment of the live mammalian eye. Our goal is to uncover the fundamental mechanisms of corneal regeneration that will unlock the full potential of stem cells for treating corneal disease.